Sybr green real time pcr master mix

Real time PCR (RT-PCR) assay was conducted to assess expression of alpha hemolysin (hla) under SNPs treatment. RNA isolation and cDNA synthesis were conducted following general procedure. Primer pair sequences for real time PCR of the virulence factor are 5′-TGAATCCTGTCGCTAATG-3′ as the forward primer; and 5′-TATCATCCGACCTTTCACT-3′ as the reverse primer (15). 16s RNA was used as housekeeping (reference) gene and internal control in RT-PCR assay. Forward and reverse sequences of the reference gene were 5′-CGTGCTACAATGGACAATACAAA-3′ and 5′-ATCTACGATTACTAGCGATTCCA-3′, respectively ^{15 (link)}. Expression of the virulence gene (hla) was quantitatively analyzed using a RT-PCR system (BioRad). RT-PCR was carried out in a 20 μl reaction volume containing 0.5 μM of each primer and 10 μl of SYBR green real time PCR master mix (Genet Bio, South Korea). Quantitative RT-PCR experiments were performed in duplicate for each sample. The RT-PCR data were analyzed by the ΔΔCt method as described by Xiang et al^{15 (link)}.